Many viruses suppress host antiviral responses through the global inhibition of host gene expression, often referred to as "host shut-off". Vesicular stomatitis virus (VSV), the prototype rhabdovirus, is a well-established example of a virus that suppresses antiviral responses through the global inhibition of host gene expression. The host shut-off in VSV-infected cells is extensive, resulting in the inhibition of host transcription, nuclear-cytoplasmic transport of host mRNA, and translation of host proteins. The work of our laboratory and others has shown that the VSV matrix (M) protein is the major inhibitor of host gene expression in VSV-infected cells, and that the ability of M protein to inhibit host gene expression is genetically separable from its function in virus assembly. The goal of this project is to determine how a relatively small (229 a.a.) protein can have such a profound effect on host gene expression. The prevailing view in the literature is that M protein blocks nuclear-cytoplasmic transport by binding the host nuclear factor Rae1 and that the inhibition of host transcription and translation are downstream effects of the inhibition of nuclear-cytoplasmic transport. However, more recent data from our laboratory and others are inconsistent with this view. Rather than a primary effect on transport, we propose an alternative model in which the M protein-Rae1 complex is an inhibitor of host transcription, and that the blocks in transport and translation of cellular mRNAs are downstream effects of this inhibition. Aim 1 is to determine the role of Rae1 in the M protein-induced inhibition of host transcription. In Aim 1a, we will determine the ability of M protein to inhibit host transcription in cells altered in their Rae1 expression by RNA silencing or overexpression of Rae1. In Aim 1b, we will determine whether formation of the M protein-Rae1 complex involves post-translational modification of Rae1 and/or formation of a complex with other host cellular factors. Specific Aim 2 is to determine the role of Rae1 in nuclear-cytoplasmic transport in VSV-infected cells. Specific Aim 3 is to determine the mechanism of inhibition of host translation in VSV-infected cells. We have shown that the translation apparatus in VSV-infected cells is altered such that new mRNAs are preferentially translated and pre-existing host mRNAs are redistributed from actively translating polysomes to translationally inactive mRNPs. Aim 3a will test the hypothesis that the inhibition of host translation is a downstream effect of the inhibition of host transcription and mRNA transport. In Aim 3b, the mechanism of translational silencing of host mRNAs will be determined by purifying the translationally inactive mRNPs and determining their protein composition by immunoblotting and proteomics approaches. These experiments will resolve discrepancies in the recent literature about the mechanism of action of M protein and the function of Rae1, and will address the relationship between transcription, mRNA transport, and translation in virus-infected cells. PUBLIC HEALTH RELEVANCE: Vesicular stomatitis virus (VSV) is an animal virus that has been studied for many years as a prototype for many important human viral pathogens. One of the important areas where study of VSV has yielded novel insights is the question of how viruses inhibit the expression of host genes. This inhibition is an important mechanism that allows viruses to replicate in the host. The goal of this project is to test new ideas about how VSV inhibits host gene expression. This will provide fundamental new information on the molecular basis of how viruses like VSV cause disease, and should contribute to development of novel viruses for use as vaccines and for the treatment of human cancer.